1. Field of the Invention
This invention generally relates to methods and systems for dynamic range expansion. Certain embodiments relate to methods and systems for dynamic range expansion in flow cytometry applications.
2. Description of the Related Art
The following descriptions and examples are not admitted to be prior art by virtue of their inclusion within this section.
Generally, flow cytometers can be used to provide measurements of the intensity of fluorescent light emitted by polystyrene beads, human cells, or other discrete substances due to exposure to an excitation source such as a laser as they pass linearly through a flow chamber. In some systems, there are four measurements that are performed: the level of light scattered by a particle at 90 degrees to the excitation source, two or more measurements of fluorescence used to determine the particle “identity,” and an additional fluorescence measurement typically used to determine and/or quantify a surface chemical reaction of interest. Each of the fluorescent measurements is typically made at a different wavelength.
The fluorescence measurement of the surface chemical reaction is typically quantified by optically projecting an image of the particle as it passes through an illumination zone of the excitation source on the photosensitive area of a photomultiplier tube (PMT) or another photosensitive detector. The output of the detector is a current pulse, which is then conditioned by analog electronics and digitized by an analog to digital (A/D) converter. The resultant digital values obtained from the A/D converter may be further conditioned in the digital domain by a digital signal processing (DSP) algorithm. The end product per particle is a single integer value, which is proportional to the chemical reaction on the surface of the particle. The fluorescent measurement(s) related to the particle identity may be performed in a similar manner. Alternatively, the integer values of the fluorescence emitted by a particle corresponding to the particle identity may be used in a different manner to determine the particle identity (e.g., by a ratio of the integer values, etc.).
The dynamic range (DR) of a flow cytometry system as described above may be generally defined as the ratio of the measurable maximum fluorescence level to measurable minimum fluorescence level. In this manner, the higher the DR, the more useful the system is at discriminating the level of chemical reaction and/or the particle identity.
The DR of currently available flow cytometers is limited by the DR of each individual element in the system (e.g., the major components including the photosensitive detector, analog electronics, and A/D converter). Typically, the photonic nature of light and noise inherent to the detector's amplification method define the detection limit at the low end of the scale, and the analog electronics and A/D converter constrain the maximum measurable fluorescence level. With commonly available off-the-shelf linear components, the useful dynamic range of flow cytometers is limited to approximately 4 decades (1 to 10,000). Usually a flow cytometry system is designed and calibrated to discern the smallest possible fluorescent signal level from the particles thereby sacrificing the ability to measure the very brightest levels of fluorescence due to the DR limits of the system.
In U.S. Pat. No. 5,367,474 to Auer et al., which is incorporated by reference as if fully set forth herein, a method to increase the DR of a flow cytometer is shown, which uses an electrical gain stage inserted between the first electrical amplifier and subsequent processing circuitry. A bypass path around the amplifier is also provided. For small signal inputs, the additional amplifier stage is used to increase the small signal, while the bypass path can be selected for signals that are already large.
This technique, while seemingly adequate to cover both small and large signal ranges, is disadvantageous in that the electrical gain stage, when inserted in the signal path, adds noise to the small signal level. It is known to those skilled in the art of flow cytometer design that the best signal-to-noise ratio occurs when the maximum electrical system gain occurs in the first circuitry stages. Thus, the bias on the photomultiplier tube, which determines its photon to electron gain factor, and is the actual first gain stage, should be maximized, and subsequent gain stages minimized.
Accordingly, it would be desirable to increase the dynamic range of a measurement system such as a flow cytometer in the first gain stage to produce the maximum signal-to-noise ratio without adding noise to small signal levels.